cAMP-mediated signaling regulates a myriad of important biological processes under physiological conditions and disease states, including diabetes, heart failure and cancer. In eukaryotic cells, the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, the classic protein kinase A/cAMP-dependent protein kinase (PKA/cAPK) and the recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (Epac/cAMP-GEF). The existence of two ubiquitously expressed cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner. However, little is known about the mechanism of Epac activation. The objective of this proposal is to fill the gap in our current knowledge by mapping the conformational changes associated with Epac activation. Specifically, we have planned experiments with the following Specific Aims: 1) To determine the specific residues important for Epac activation by site-directed mutagenesis; 2) to delineate the conformational changes associated with cAMP binding and Epac activation and to determine the protein interface between Epac and it's downstream effector, Rap1, using enhanced deuterium exchange-mass spectrometry (DXMS) and small-angle X-ray scattering; and 3) to solve the crystal structure of Epac2-cAMP using X-ray crystallography. The long-term goals of our research are to understand the physiological functions and mechanisms of Epac regulation. Accomplishing the proposed research in this application will significantly move the field forward towards these goals. Furthermore, the medical and pharmacological implications of this research program are also far-reaching. A better understanding of cAMP- mediated signal transduction could potentially lead to the identification of novel mechanism-based therapeutic strategies specifically targeting the cAMP-signaling components.